The object of the present investigation was to determine whether insulin secreted by the endocrine pancreas and carried in the insulo-acinar portal system has a direct effect on pancreatic enzyme secretion. For this purpose, the isolated rat pancreas was perfused in a nonrecirculating system. The perfusate contained 3 mM glucose, and either caerulein or vaso-active intestinal polypeptide was used to stimulate exocrine secretion. The amount of insulin reaching the exocrine pancreas was reduced by two different experimental procedures. In the first, use was made of streptozotocin-diabetic rats treated with insulin in vivo. Treatment was such that the contents of amylase and lipase, vastly altered in the untreated diabetic state, were normalized before the perfusion studies. In the second procedure, insulin reaching the exocrine pancreas was reduced by antiinsulin serum in the perfusate. In these procedures, the reduced insulin bioavailability was associated with a reduction in caerulein- and vasoactive intestinal polypeptide-stimulated enzyme release, which was shown as a reduction of maximum responsiveness to caerulein without alteration of sensitivity. By contrast, in dispersed pancreatic acini where the insulo-acinar axis was completely disrupted, amylase secretion from diabetic and nondiabetic tissue was identical over a wide range of caerulein concentrations, showing that the secretory defect seen in the perfusion studies was not inherent to the exocrine tissue. The results show that basal insulin secretion has a direct effect on pancreatic enzyme output and that the insulo-acinar axis may play an important role in the regulation of acinar cell function.